The post-translational modifications of proteins with proteins of the ubiquitin family are highly dynamic processes that increase the potential of the cell to adapt to multiple physiological or pathological situations (Hayashi et al., 2002; Seufert et al., 1995). Modification of proteins by the small ubiquitin modifier (SUMO) results in very diverse outcomes. The first identified targets of SUMO, the oncogenic protein PML (promyelocytic leukemia) and the nucleocytoplasmatic transport protein RanGAP1 (RanGTPase-activating1), allowed to connect SUMO functions to nuclear localization (Matunis et al., 1996; Lapenta et al., 1997; Mahajan et al. 1997; Wilkinson and Henley, 2010). The identification and characterization of hundreds of SUMO substrates suggest the role of SUMO as regulator of protein activity and stability (Seeler and Dejean, 2003). Downstream consequences are mediated, at least in part, by effectors that contain SUMO-interacting motifs or SIMs.
SUMO proteins only have a 20% of identity with ubiquitin, however, have a similar three-dimensional structure (Kerscher et al., 2006) (FIG. 1). Three forms of SUMO (SUMO-1, SUMO-2 and SUMO-3) are ubiquitously expressed and migrate with an apparent molecular weight of around 10 kDa in a denaturing polyacrylamide gel. SUMO-1 shares only 50% of identity with SUMO-2 and SUMO-3, but SUMO-2 and SUMO-3 are 97% identical, and form a distinct subfamily known as SUMO 2/3 (Tatham et al., 2001).
As it occurs with ubiquitin, SUMO molecules are generated through the processing of a high molecular weight precursor, the cleavage of which exposes the double glycine signature that is involved in their conjugation to protein substrates. A thiol-esther cascade of 3 reactions mediates the conjugation of SUMO molecules (FIG. 2). A single SUMO activating enzyme (SAE) or E1, activate all SUMO molecules that are conjugated by the E2 Ubc9 (Hay, 2005; Wilkinson and Henley, 2010).
This process is facilitated by a specific SUMO E3 ligase. SUMO E3 ligases, such as members of the PIAS (protein inhibitor of activated STAT) family and RanBP3 (Ran-binding protein 3), contain a SP-RING (Really Interesting New Gene) motif which is essential for their function (Kahyo et al., 2001 Schmidt and Muller, 2002). A distinct group of SUMO-ligases is composed by Polycom protein (Pc2) which is associated to gene silencing (Kagey et al., 2003). SUMOylation is a highly reversible modification that is mediated by SUMO specific cystein proteases (SUSPs or SENPs). In mammals 6 SENPs were initially reported, of which SENP1-SENP3 and SENP5-SENP7 are specific for SUMO proteins. The localization of SENPs and specificity for SUMO-isoforms determines their action (Yeh, 2009). The initial mapping of SUMO modification lysine residues allowed the identification of a SUMO consensus site composed by ψKxE, in which Ψ is a large hydrophobic aminoacid and x any aminoacid (Rodriguez et al., 2001; Xu et al., 2008). Such consensus was found in RanGAP1, PML, p53 and IκBα, among other proteins and interacts directly with Ubc9 (Sampson et al., 2001; Wilkinson and Henley, 2010). The target lysine enters into the catalytic pocket of Ubc9 whereas the hydrophobic and acidic residues interact with the surface of this E2 (Bernier-Villamor et al., 2002). Although, not all SUMOylation sites fit with the canonical consensus, modification will only occur if the consensus presents an unstructured region or a well exposed substrate surface (Geiss-Friedlander and Melchior, 2007).
The stimulation of SUMO proteins to their substrates could be reached by cellular stress, such heat shock. Golebiowski and colleagues (Golebiowski, 2009) described a quantitative change in global SUMOylation profiles during heat shock, because previous experiments demonstrated a rapid and reversible increase in SUMO-2 and SUMO-3 conjugates when cells were shifted from the normal growth temperature of 37° C. to the stressful 43° C. (Saitoh and Hinchey, 2000).
The consequences of the SUMOylation of a new protein target are difficult to predict as the changes of conformation, creation or masking interaction surfaces may affect activity, stability and localization of modified proteins. SUMOylated proteins are recognized by SUMO interacting motifs (SIMs) present in a large diversity of proteins including promyelocytic leukemia (PML), death domain-associated protein (Daxx), ubiquitin-like modifier activating enzyme 2 (UBA2), protein inhibitors of activated STAT (PIAS) and RING finger protein 4 (RNF4) ligases (Geiss Friedlander and Melchior 2007). SIMs contain a hydrophobic core flanked by acidic (E/D) and a serine residues (Song et al., 2004; Hecker et al., 2006). SIM motifs form a β-strand that bind in parallel or anti-parallel orientation between the α-helix and a βstrand of SUMO (FIG. 3).
The affinity of SIM and SUMO usually is low, around the high micromolar range, which is normally due to the reduced surface of interaction. To increase such affinity, proteins such as the SUMO-dependent ubiquitin ligase RNF4 contain 4 SIM domains. SIM2 and SIM3 appear to play a more important role in the capture of SUMOylated proteins (Tatham et al., 2008). In this way SUMOylated PML is targeted to degradation by the ubiquitin-proteasome system, providing the first molecular evidence about how SUMO molecules can contribute to regulate protein stability. Affinity columns containing 4 SIMs derived form natural occurring fragment 32-133 of RNF4 have been published (Bruderer R et al. 2011) and commercialized by BIOMOL (Affinity research).